Shim Jae Youn, Lee Byung Hun, Park Hye Yoon
Department of Physics and Astronomy, Seoul National University, Seoul, South Korea.
The Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Methods Mol Biol. 2019;2038:47-61. doi: 10.1007/978-1-4939-9674-2_4.
Transcription and post-transcriptional regulations are critical in gene expression. To study the spatiotemporal regulation of RNA inside a cell, techniques for high-resolution imaging of RNA have been developed. In this chapter, we describe RNA fluorescent labeling methods using MS2 and PP7 systems to detect single RNA molecules in live neurons. We use hippocampal neurons cultured from knock-in mouse models in which β-actin or Arc mRNAs are tagged with MS2 or PP7 stem-loops. Adeno-associated virus (AAV) or lentiviral vectors are used to express MS2 or PP7 capsid proteins fused with GFP in those neurons. Then, GFP-labeled RNAs in live neurons can be detected by epifluorescence microscopy, and their moving pathways can be analyzed using single-particle tracking software. For these processes, we introduce protocols for neuron culture, transfection, imaging, and particle tracking methods.
转录及转录后调控在基因表达中至关重要。为了研究细胞内RNA的时空调控，已开发出用于RNA高分辨率成像的技术。在本章中，我们描述了使用MS2和PP7系统的RNA荧光标记方法，以检测活神经元中的单个RNA分子。我们使用从敲入小鼠模型培养的海马神经元，其中β-肌动蛋白或Arc mRNA用MS2或PP7茎环进行标记。腺相关病毒（AAV）或慢病毒载体用于在这些神经元中表达与GFP融合的MS2或PP7衣壳蛋白。然后，可以通过落射荧光显微镜检测活神经元中GFP标记的RNA，并使用单粒子跟踪软件分析其移动路径。对于这些过程，我们介绍了神经元培养、转染、成像和粒子跟踪方法的方案。
